I followed the standard SYBR Green Protocol for doing a qPCR. For which I used

10 uL of 1X SYBR Green Master Mix

Forward Primer and Reverse Primer (each at a final conc. = 8.5 uM)

Template (unknown concentration from an aptamer selection)

Made to 20 uL with water.

Performed the PCR amplification using

95 C Initial denaturation

95, 68, 72 - cycled 35 times.

But, one set of sample from my aptamer selection (probably high GC content) did not amplify at all. I checked on an agarose gel too.

Important Notes:

My primer conc. is intentionally high - I have always amplified with such concentrations and it works perfectly using Vent Polymerase.

My annealing temperature is also high at 68 C because my aptamer sequences tend to be GC Rich.

Using Vent polymerase my amplification is very strong

On reading notes from vendors, I found out that the polymerase in the mastermix to be some variant of Taq Polymerase[1][3] (varies from vendor to vendor) and also it is known to poorly amplify GC Rich Sequences[2].